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Blood, Vol. 108, Issue 12, 3928-3937, December 1, 2006
SOCS up-regulation mobilizes autologous stem cells through CXCR4 blockade
Blood Pello et al.
108: 3928
Supplemental materials for: Pello et al, Vol 108, Issue 12, 3928-3937
Files in this Data Supplement:
- Figure S1. Design of lentiviral vectors (JPEG, 26.3 KB)
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pLVTHM-GFP, pLVTHM-SOCS1-GFP, and pLVTHM-SOCS3-GFP lentiviral vectors containing SOCS genes were constructed from controllable siRNA (Tronolab, Lausanne, Switzerland) by replacing the H1 Pol III promoter with the cytomegalovirus promoter (CMV) and by adding a BGH polyadenylation sequence (BGH/pA); when necessary, CDA for SOCS-1 or SOCS-3 was cloned between the CMV and BGH/pA sequences. The remaining elements of the original construct were maintained, and include transgenes expressing elongation factor 1-alpha (EF-1 ), a central polypurine tract (cPPT), the green fluorescent protein (GFP) marker, the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE), and a viral long-terminal repeat (LTR) with a self-inactivating (SIN) element. The tetracycline operator element (tetO) is induced by tetracycline binding to the tetracycline repressor fused to the KRAB repression domain of human Kox-1 (tTR-KRAB) factor in the pLV-tTRKRAB-dsRED vector.
- Figure S2. Y chromosome FISH (JPEG, 12.7 KB)
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Lyn+ cells from BM of female mice reconstituted by intrafemoral injection of whole peripheral blood from male bGHTg mice were hybridized with a Cy3-labeled probe for the Y chromosome (red). Nuclei were counterstained with DAPI (blue). The presence of the Y chromosome in most cells demonstrates that transplanted BM reconstituted the irradiated recipients.
- Figure S3. SOCS expression did not modify CXCL12 levels in BM (JPEG, 33 KB)
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Quantitative RT-PCR analysis of CXCL12 mRNA from BM cells of mice reconstituted with tetO/SOCS1- and tetO/SOCS3-transduced cells, before and after doxycycline treatment. Results are expressed as x-fold increase (“Materials and methods”). The mean ± SD is shown for 3 independent experiments.
- Figure S4. Proliferation of BM cells from mice reconstituted with lentivirus-infected cells (JPEG, 39.9 KB)
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BrdU incorporation in BM cells from mice reconstituted with tetO/empty-, tetO/SOCS1-, and tetO/SOCS3-transduced cells, in the absence of doxycycline treatment. Histograms correspond to representative animals (n = 4).
- Figure S5. Proliferation of lentivirus-infected Lin– cells cocultured on a stroma cell line monolayer (JPEG, 50.1 KB)
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BrdU incorporation in pLVTHM-tetO/SOCS1-, tetO/SOCS3-, or tetO/mock-transduced Lin– cells plated on confluent MS-5 monolayers and cocultured for 20 or 40 hours, alone or with tetracycline. Histograms correspond to a representative experiment (n = 8).
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